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WO2025068313A1 - Procédé de diagnostic précoce, de prédiction précoce, de surveillance ou de prédiction de la gravité d'une fonction de greffe réduite chez un patient de transplantation rénale - Google Patents

Procédé de diagnostic précoce, de prédiction précoce, de surveillance ou de prédiction de la gravité d'une fonction de greffe réduite chez un patient de transplantation rénale Download PDF

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Publication number
WO2025068313A1
WO2025068313A1 PCT/EP2024/076971 EP2024076971W WO2025068313A1 WO 2025068313 A1 WO2025068313 A1 WO 2025068313A1 EP 2024076971 W EP2024076971 W EP 2024076971W WO 2025068313 A1 WO2025068313 A1 WO 2025068313A1
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graft function
prediction
patient
risk
kidney
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Christian Karl NUßHAG
Markus WEIGAND
Oliver Hartmann
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Sphingotec GmbH
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Sphingotec GmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/665—Assays involving proteins derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G01N2800/245—Transplantation related diseases, e.g. graft versus host disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • S75613WO BOEHMERT & BOEHMERT A method for early diagnosis, early prediction, monitoring or prediction of severity of reduced graft function in a kidney transplantation patient
  • Subject matter of the present invention is a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis and/ or risk and/ or the severity of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Subject matter of the present invention is a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with graft function and/ or risk and/ or the severity of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is a method patient stratification and/ or patient selection for early treatment of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and ⁇ stratifying and/ or selecting said patient for early treatment of reduced graft function in correlation of said level of Pro-Enkephalin or fragments thereof in said sample, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to the present invention, wherein said method is used for patient stratification and/ or patient selection for early treatment of reduced graft function in a kidney transplantation patient.
  • Further subject matter of the present invention is also a method for early diagnosis of graft function and/ or for early diagnosis and/ or early prediction of recovery from reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the recovery from of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is also a method for early diagnosis and/ or early prediction of immediate graft function (IGF) in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the graft function, wherein the graft function is immediate graft function (IGF) of the kidney.
  • IGF immediate graft function
  • DGF Delayed graft function
  • DGF is the term used to describe the failure of the transplanted kidney to function immediately after transplantation. It can be considered a form of acute kidney injury post- transplantation and is an important complication of kidney transplantation.
  • DGF DGF-derived neurotrophic factor
  • DGF ischemia–reperfusion injury
  • IRI ischemia–reperfusion injury
  • the source of donated kidney deceased vs. living donation
  • the quality of the donated kidney and the clinical conditions of the recipient
  • DGF predisposes the graft to both acute and chronic rejection (Boom et al.2000. Kidney Int 58: 859–866) and increases the risk of chronic allograft nephropathy and premature graft loss (Giral-Classe et al. 1998. Kidney Int 54: 972–978).
  • Kidneys for transplantation may be removed from living (relatives of recipients or unrelated donors) or deceased donors (brain-dead donors) with irreversible brain injury and persistent circulation maintained by supportive measures or donors after circulatory death. The risk of DGF is higher for deceased donors (Yasseri et al.2021. Urol.
  • ECDs expanded criteria donors
  • Kidney allografts from ECDs have two-fold increased risk of DGF, more frequent acute rejection, and lower graft function in the long-term (Port et al. 2002. Transplantation 74: 1281–1286).
  • Post-transplant renal function has historically been classified patients merely by the presence or absence of DGF. As a result, many patients may have significant injury but, by default, are considered to have “adequate” graft function if they avoid dialysis (Akkina et al. 2009. Am J Transplant 9: 1460–66).
  • SGF slow graft function
  • IGF immediate graft function
  • SGF SGF
  • SCr serum creatinin
  • Proenkephalin A is a precursor of the enkephalin family of endogenous opioids. It is a prohormone that is proteolytically processed to form several active pentapeptides like methionine-enkephalin (Met-Enk) and leucine-enkephalin (Leu-Enk) together with several other peptide fragments (enkelytin and C-terminal extended Met-Enk peptides).
  • Method-Enk methionine-enkephalin
  • Leu-Enk leucine-enkephalin
  • PENK 119-159 proenkephalin peptide 119-159
  • This peptide fragment levels in plasma/serum could serve as a surrogate measurement of systemic enkephalin synthesis, because proenkephalin is the predominant source of mature enkephalins.
  • Enkephalins are widely secreted to act on locally expressed opioid receptors, specifically the ⁇ opioid receptors. These opioid receptors are also widely expressed, with the highest density found in the kidney (Denning et al.2008. Peptides 29 (1): 83–921).
  • PENK 119-159 strongly correlates with the kidney function and measured GFR (Beunders et al. 2020 54(3): 308–314). Therefore, it was proposed as a biomarker for assessing kidney function in critically ill patients (Beunders et al.2017. Appl Lab Med 2(3): 400-412; Donato et al.2018.
  • PENK is associated with kidney function as reflected by correlations with measured GFR in both renal transplant recipients and kidney donors one year after kidney transplantation.
  • PENK was independently associated with increased risk for late graft failure in renal transplant recipients using PENK levels measured at least one year after kidney transplantation (Kienecker et al.2017. Transplantation Direct 3: e190).
  • PENK levels measured in newly transplanted patients e.g., within hours or days after kidney transplantation.
  • Subject matter of the present invention is a method for early diagnosis and/ or early prediction and/ or monitoring a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis and/ or the risk and/ or severity of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Subject matter of the present invention is a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with graft function and/ or risk and/ or the severity of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is a method patient stratification and/ or patient selection for early treatment of reduced graft function in a kidney patient transplantation comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and ⁇ stratifying and/ or selecting said patient for early treatment of reduced graft function in correlation of said level of Pro-Enkephalin or fragments thereof in said sample, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to the present invention, wherein said method is used for patient stratification and/ or patient selection for early treatment of reduced graft function in a kidney transplantation patient.
  • Further subject matter of the present invention is also a method for early diagnosis and/ or early prediction of recovery from reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the recovery from of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Further subject matter of the present invention is also a method for early diagnosis and/ or early prediction of immediate graft function (IGF) in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the graft function, wherein the graft function is immediate graft function (IGF) of the kidney.
  • IGF immediate graft function
  • Further subject matter of the present invention is a method for early treatment of reduced graft function in a kidney transplantation patient, wherein the treatment is selected from the group comprising renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive therapy and/ or adjustment of nephrotoxic drugs, wherein said patient is selected by a diagnostic method comprising the steps: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis and/or risk and/ or severity of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • a further subject matter of the present invention is a medicament for use in early treatment of reduced graft function in a kidney transplantation patient, wherein said patient is selected by a diagnostic method comprising the steps: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis of reduced graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Proenkephalin (PENK) or fragments thereof are powerful and highly significant biomarkers for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity reduced graft function in a kidney transplantation patient. Moreover, it has been shown to be especially useful for the early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of delayed graft function (DGF) in a kidney transplantation patient. It was a further surprising finding that levels of PENK or fragments thereof can be used in a kidney transplantation patient to predict short-term kidney function, especially a reduced kidney function (e.g. as reduced GFR) up to 30 days after kidney transplantation.
  • a reduced kidney function e.g. as reduced GFR
  • correlating refers to comparing the presence or level of the marker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition (e.g. DGF or SGF or reduced kidney function).
  • a marker level in a patient sample can be compared to a level known to be associated with a specific diagnosis.
  • the sample's marker level is said to have been correlated with a diagnosis; that is, the skilled artisan can use the marker level to determine whether the patient suffers from a specific type of a disease and respond accordingly.
  • the sample ⁇ s marker level can be compared to a marker level known to be associated with the prediction of a disease or an outcome of a disease (e.g. the development of a disease or condition, which may be for example DGF or SGF, the severity of a disease or condition or an improvement or worsening of a disease or condition, e.g. kidney function).
  • a disease or condition which may be for example DGF or SGF
  • the severity of a disease or condition or an improvement or worsening of a disease or condition e.g. kidney function.
  • kidney function e.g. the expression of a disease or condition.
  • the term "patient” as used herein refers to a living human or non-human organism.
  • the patient is a human kidney transplantation patient.
  • the term facedkidney transplantation patient“ means a patient who is planned for kidney transplantation, a patient who is under kidney transplantation or a patient who already received kidney transplantation.
  • the term “child” as used herein refers to a subject that is at the age of 18 years or below, more preferred at the age of 14 years or below, even more preferred at the age of 12 years or below, even more preferred at the age of 8 years or below, even more preferred at the age of 5 years or below, even more preferred at the age of 2 years or below, most preferred at the age of one year or below.
  • the term “elevated level” means a level above a certain (predetermined) threshold level.
  • the term “elevated” level may mean a level above a value that is regarded as being a reference and/ or threshold level.
  • the term “diagnosing” in the context of the present invention relates to the recognition and (early) detection of a disease or clinical condition in a subject and may also comprise differential diagnosis.
  • the term “early diagnosis” in the context of the present invention relates to the timepoint of the diagnosis of a disease or clinical condition in a subject which is before the usual time that the disease or clinical condition is diagnosed by gold standard methods.
  • “early diagnosis” means within 96 hours, preferably within 72 hours, more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • said early diagnosis means within 12 to 48 hours after kidney transplantation.
  • said early diagnosis means within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.
  • “early diagnosis” is the “early diagnosis” of graft function of the kidney in a kidney transplantation patient.
  • “early diagnosis” is the “early diagnosis” of reduced graft function of the kidney in a kidney transplantation patient.
  • the term trainedprediction“ in the context of the present invention denotes a prediction of how a patient’s medical condition will progress. This may include an estimation of the chance of recovery or the chance (risk) of an adverse outcome (e.g., SGF or DGF, reduction in kidney function) for said patient.
  • age prediction in the context of the present invention relates to the timepoint of the prediction of how a patient’s medical condition will progress which is before the usual time that a particular event happens (e.g., the occurrence of first symptoms of a disease).
  • “early prediction” means within 96 hours, preferably within 72 hours, more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • said early prediction means within 12 to 48 hours after kidney transplantation.
  • said early prediction means within 12 to 24 hours after kidney transplantation.
  • said early prediction means within 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13 or 12 hours.
  • the term “early” in the context of diagnosis and/ or prediction means that at the timepoint of diagnosis of reduced graft function and/ or prediction of a risk of reduced graft function and/ or prediction of the severity of reduced graft function using Proenkephalin or fragments thereof, the biomarker serum creatinine (SCr) does not diagnose reduced graft function and/ or does not predict a risk of reduced graft function and/ or does not predict the severity of reduced graft function in a kidney transplantation patient.
  • SCr biomarker serum creatinine
  • the term “early” in the context of diagnosis and/ or prediction means that at the timepoint of diagnosis of reduced graft function and/ or prediction of a risk of reduced graft function and/ or prediction of the severity of reduced graft function using Proenkephalin or fragments thereof (i) the level of serum creatinine (SCr) is still above a predetermined threshold level and/ or (ii) the relative change of SCr is a decrease of less than 50 %.
  • said predetermined threshold level of SCr is in the range between 1.5 and 4 mg/dL, more preferred in the range between 1.5 and 3 mg/dL, more preferred in the range between 1.5 and 2.5 mg/dL, most preferred said predetermined threshold is 2 mg/dL.
  • the term “early” in the context of diagnosis and/ or prediction means within 96 hours, preferably within 72 hours, more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours and wherein (i) said level of SCr is above a predetermined threshold level and/ or (ii) the relative change of SCr is a decrease of less than 50 % in said kidney transplantation patient.
  • the term “early” in the context of diagnosis and/ or prediction means that at a specific timepoint said level or relative change of Proenkephalin or fragments thereof diagnoses or predicts that said kidney transplantation patient does not have or will not develop reduced graft function but said level or relative change of SCr is still above a predetermined threshold or the decrease is less than 50%.
  • kidney transplantation patient might be falsely diagnosed as having reduced graft function if the level or relative change of Proenkephalin or fragments thereof would not be considered within the first days after kidney transplantation.
  • the prediction of kidney function in a kidney transplantation patient means prediction within 12 months, more preferred within 9 months, more preferred within 6 months, more preferred within 3 months, more preferred within 1 month, most preferred within 14 days.
  • the prediction of kidney function in a kidney transplantation patient means short-term prediction within 1 month, more preferred within 28 days, even more preferred within 21 days, most preferred within 14 days.
  • the term “ambulmonitoring“ refers to controlling the development (detection of any changes) of a disease and or pathophysiological condition of a patient, e.g. risk or severity of a disease or condition or response to a therapy.
  • Said patient monitoring or follow-up will be up to 28 days after kidney transplantation or until the graft function of the transplanted kidney is restored.
  • Said follow-up measurements may be performed up to 7 days, preferably up to 14 days, more preferred up to 21 days, most preferred up to 28 days. In one embodiment said follow-up measurements may be performed until the kidney function is reserved.
  • the term "monitoring the success of a therapy or intervention" in the context of the present invention refers to the control and/or adjustment of a therapeutic treatment of said patient.
  • Predicting or monitoring the success of a therapy or intervention may be e.g., the prediction or monitoring the success of renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive drugs and/ or adjustment of nephrotoxic drugs using measurement of Pro-Enkephalin (PENK) or fragments thereof in a patient diagnosed and/ or being at risk of reduced graft function after kidney transplantation.
  • Predicting or monitoring the success of a therapy or intervention may be e.g. the prediction or monitoring of the recovery of renal function in patients at risk of reduced graft function after kidney transplantation prior to and after renal replacement therapy and/or administration of a medicament and/ or adjustment of immunosuppressive drugs and/ or adjustment of nephrotoxic drugs using measurement of PENK or fragments thereof.
  • Before transplantation is defined as any time up to 7 days before transplantation procedure starts.
  • said sample taken before transplantation is obtained within 7 days, preferably 6 days, more preferred within 5 days, even more preferred within 4 days, even more preferred within 3 days, even more preferred within 48 hours, more preferred within 24 hours, preferably within 12 hours most preferred within 6 hours before transplantation procedure starts.
  • the normal hospital procedure would be to take said sample within 24 hours before transplantation procedure starts.
  • One embodiment of the invention is a method for predicting or monitoring the success of a therapy or intervention in a patient identified as having and/ or being at risk of reduced graft function after kidney transplantation, wherein a worsening or recovery of renal function prior to and/ or after therapy or intervention is predicted or monitored.
  • DGF delayed graft function
  • SGF slow graft function
  • IGF immediate graft function
  • GFR glomerular filtration rate
  • GFR is equal to the total of the filtration rates of the functioning nephrons in the kidney and is considered the most useful index of kidney function in health and disease.
  • the GFR is typically recorded in units of volume per time, e.g., milliliters per minute (mL/min). Glomerular filtration cannot be measured directly in humans; thus “true” GFR cannot be known with certainty. However, GFR can be assessed from clearance measurements (measured GFR [mGFR]) or serum levels of endogenous filtration markers (estimated GFR [eGFR]). The GFR can be measured by injecting inulin or the inulin-analog sinistrin into the blood stream (“measured GFR”).
  • kidney function is the current "gold standard" for comparison with other means of estimating glomerular filtration rate.
  • the contrast agents Iohexol and Iothalamate have become more popular alternatives to determine GFR and are considered to show sufficient accuracy to determine GFR (Soveri et al.2014. Am J Kidney Dis.64(3):411-24).
  • the creatinine clearance rate (CCr or CrCl) is the volume of blood plasma that is cleared of creatinine per unit time and is a useful measure for approximating the GFR. Creatinine clearance exceeds GFR due to creatinine secretion, which can be blocked by cimetidine.
  • GFR and CCr may be accurately calculated by comparative measurements of substances in the blood and urine or estimated by formulas using just a blood test result (eGFR and eCCr). The results of these tests are used to assess the excretory function of the kidneys.
  • eGFR blood test result
  • mGFR measured GFR
  • Estimated GFR may be calculated for example using CKD-EPI creatinine equation, CKD-EPI cystatin C equation or CKD-EPI creatinine cystatin C equation, the Modification of Diet in Renal Disease (MDRD) study equation, and Cockcroft-Gault equation (Santos and Martins 2015. World J Nephrol 4(3): 345-353).
  • kidney disease The severity of chronic kidney disease (CKD) is described by six stages; the most severe three are defined by the MDRD-eGFR value, and first three also depend on whether there is other evidence of kidney disease (e.g., proteinuria): 0) Normal kidney function – GFR above 90 (mL/min)/(1.73 m2) and no proteinuria 1) CKD1 – GFR above 90 (mL/min)/(1.73 m2) with evidence of kidney damage 2) CKD2 (mild) – GFR of 60 to 89 (mL/min)/(1.73 m2) with evidence of kidney damage 3) CKD3 (moderate) – GFR of 30 to 59 (mL/min)/(1.73 m2) 4) CKD4 (severe) – GFR of 15 to 29 (mL/min)/(1.73 m2) 5) CKD5 kidney failure – GFR less than 15 (mL/min)/(1.73 m2).
  • CKD4 severe
  • kidney function in the context of the present invention may be determined by glomerular filtration rate (GFR), creatinine clearance rate (CCr), serum creatinine (SCr), serum cystatin C (CyC), urinalysis, blood urea nitrogen or urine output.
  • GFR may be selected from estimated GFR (eGFR), true GFR or measured GFR (mGFR).
  • eGFR estimated GFR
  • mGFR measured GFR
  • a reduction of kidney function is predicted in said patient.
  • said reduction of kidney function is determined by GFR.
  • a reduction of GFR below 60, preferably below 45, preferably below 30, most preferred below 15 is predicted.
  • Proenkephalin or fragments thereof are diagnostic and/ or predictive of whether the patient is either having or developing slow graft function or delayed graft function.
  • the level of Proenkephalin or fragments thereof can distinguish between a patient that has or will develop SGF and a patient that has or will develop DGF.
  • the level of Proenkephalin or fragments thereof can distinguish between a patient that has or will develop SGF and a patient that has or will develop DGF, wherein (i) if said level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient is elevated above a predetermined threshold level, said patient has or is at risk of DGF, whereas (ii) if said level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient is below a predetermined threshold level, said patient has or is at risk of SGF.
  • said predetermined threshold level to distinguish between a patient that has or will develop SGF and a patient that has or will develop DGF is in the range between 100 and 500 pmol /L, more preferred in the range between 150 and 400 pmol/L, most preferred in the range between 200 and 350 pmol/L.
  • said patient will receive renal replacement therapy if delayed graft function is diagnosed and/ or predicted and will not receive renal replacement therapy if slow graft function is diagnosed or predicted. Severity of delayed graft function is defined as the number of days the patient needs renal replacement therapy.
  • a bodily fluid may be selected from the group comprising blood, serum, plasma, urine, cerebrospinal fluid (CSF), and saliva. In one embodiment of the invention the bodily fluid is selected from the group comprising whole blood, blood plasma, and blood serum.
  • Pro-Enkephalin or fragments thereof are diagnostic and/ or predictive for reduced graft function and/ or predictive for the severity of reduced graft function in a kidney transplantation patient, wherein a sample from said patient is taken (a) at least once before and at least once after or (b) at least twice after kidney transplantation and wherein (i) an elevated level above a certain predetermined threshold or (ii) a relative change between the level of Pro-Enkephalin or fragments thereof in the samples either (a) taken before and after or twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%, is diagnostic and/ or predictive for reduced graft function and/ or predictive for the severity of reduced graft function in said patient.
  • Pro-Enkephalin or fragments thereof are diagnostic and/ or predictive for delayed graft function and/ or predictive for the severity of delayed graft function in a kidney transplantation patient, wherein a sample of bodily fluid from said patient is taken at least once before and at least once after or at least twice after kidney transplantation and wherein (i) an elevated level above a certain predetermined threshold or (ii) a relative change between the level of Pro-Enkephalin or fragments thereof in the samples either taken before and after or twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%, is diagnostic and/ or predictive for delayed graft function and/ or predictive for the severity of delayed graft function in said patient.
  • the level of Pro-Enkephalin or fragments thereof or (ii) a relative change of Pro-Enkephalin or fragments thereof correlates with the improvement or worsening of kidney function, wherein (i) a decrease of the level of Pro- Enkephalin or fragments thereof or (ii) a decrease of the relative change of Pro-Enkephalin or fragments thereof of more than 50% correlates with the improvement of kidney function and wherein (i) an increase of Pro-Enkephalin or fragments thereof or (ii) an either a decrease of less than 50% or an increase of the relative change of Pro-Enkephalin or fragments thereof correlates with a worsening of the kidney function in a patient that has been diagnosed and/ or predicted to be at risk of reduced graft function, in particular slow graft function or delayed graft function.
  • the level of Pro-Enkephalin or fragments thereof or (ii) a relative change of Pro-Enkephalin or fragments thereof correlates with the success of the therapy or intervention, wherein (i) a decrease of the level of Pro-Enkephalin or fragments thereof or (ii) a decrease of the relative change of Pro-Enkephalin or fragments thereof of more than 50% correlates with a successful therapy or intervention and wherein (i) an increase of Pro-Enkephalin or fragments thereof or (ii) either a decrease of less than 50% or an increase of the relative change of Pro-Enkephalin or fragments thereof correlates with an unsuccessful therapy or intervention in a patient that has been diagnosed and/ or predicted to be at risk of reduced graft function, in particular slow graft function or delayed graft function.
  • said therapy or intervention is renal replacement therapy, that is stopped and/ or withheld if said level of Proenkephalin or fragments thereof is below a predetermined threshold or the decrease of the relative change of Pro-Enkephalin or fragments thereof is more than 50%.
  • a relative change of the level of a biomarker e.g., the biomarker Proenkephalin or fragments thereof or the biomarker serum creatinine
  • Pro-Enkephalin or fragments thereof are superior in comparison to other markers for early diagnosis and/ or early prediction of the risk and/ or monitoring the risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient (e.g., blood creatinine, creatinine clearance). Superiority means higher specificity, higher sensitivity, better correlation to clinical endpoints and at an earlier timepoint. Kidney function may be measured by GFR, creatinine clearance, SCr, cystatin C, urinalysis, blood urea nitrogen or urine output.
  • Renal replacement therapy replaces the normal blood-filtering function of the kidneys.
  • Renal replacement therapy may refer to dialysis (e.g. hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.
  • the hemodialysis, hemofiltration, and hemodiafiltration may be continuous or intermittent and can use an arteriovenous route (in which blood leaves from an artery and returns via a vein) or a venovenous route (in which blood leaves from a vein and returns via a vein). This results in various types of RRT.
  • the renal replacement therapy may be selected from the group of, but not limited to continuous renal replacement therapy (CRRT), continuous hemodialysis (CHD), continuous arteriovenous hemodialysis (CAVHD), continuous venovenous hemodialysis (CVVHD), continuous hemofiltration (CHF), continuous arteriovenous hemofiltration (CAVH or CAVHF), continuous venovenous hemofiltration (CVVH or CVVHF), continuous hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venovenous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis (IHD), intermittent venovenous hemodialysis (IVVHD), intermittent hemofiltration (IHF), intermittent venovenous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF) and intermittent venovenous hemodiafiltration (IVVHDF).
  • CRRT continuous renal replacement therapy
  • CHD continuous hemodialysis
  • CAVHD continuous arteriovenous
  • renal replacement therapy is selected from the group comprising dialysis (hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.
  • patients being at risk of reduced graft function after kidney transplantation may be administered a medicament selected from the group comprising recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, C1 esterase inhibitor.
  • Immunosuppressive therapeutic drugs are selected from the group comprising interleukin 2 receptor antagonists, calcineurin inhibitors (e.g., cyclosporine A, tacrolimus), mammalian target of rapamycin inhibitor, corticosteroids (e.g., prednisolone), mycophenolate, sirolimus, azathioprine.
  • interleukin 2 receptor antagonists e.g., interleukin 2 receptor antagonists
  • calcineurin inhibitors e.g., cyclosporine A, tacrolimus
  • mammalian target of rapamycin inhibitor e.g., corticosteroids (e.g., prednisolone), mycophenolate, sirolimus, azathioprine.
  • Nephrotoxic medications may be selected from the group comprising calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), pain medications (e.g., Nonsteroidal anti- inflammatory drugs (NSAIDs) as ibuprofen, aspirin), anti-microbials (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g,. cisplatin).
  • calcineurin inhibitors for immunosuppression e.g., cyclosporine A, tacrolimus
  • pain medications e.g., Nonsteroidal anti- inflammatory drugs (NSAIDs) as ibuprofen, aspirin
  • anti-microbials e.g
  • said adjustment of nephrotoxic medication and/ or adjustment of immunosuppressive therapy of a patient can involve initiation and/ or a change and/ or withdrawl of said nephrotoxic medication and/ or immunosuppressive therapeutic drugs.
  • Said adjustment of nephrotoxic medication and/ or adjustment immunosuppressive therapeutic drugs may be a change in the dose, the administration route or regime or other parameters of nephrotoxic medication treatment.
  • an adjustment in the treatment with nephrotoxic medication and/ or immunosuppressive therapeutic drugs may also and potentially additionally relate to a change in the one or more nephrotoxic agents and/ or immunosuppressive therapeutic drugs used for treating the patient.
  • a change can therefore relate to the replacement of one or more nephrotoxic medicaments and/ or immunosuppressive therapeutic drugs by one or more other agents.
  • said nephrotoxic medication and/ or immunosuppressive therapeutic drug is withdrawn from the patient.
  • Pro-Enkephalin, proenkephalin and PENK are used synonymously throughout the specification.
  • Pro-Enkephalin has the following sequence: SEQ ID NO.1 (Pro-Enkephalin 1-243) ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTL RENSKPEESHLLAKRYGGFMKRYGGFMKKMDELYPMEPEEEANGSEILAKRYGGFMK KDAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVSKRYGGFMRGLKRSPQL EDEAKELQKRYGGFMRRVGRPEWWMDYQKRYGGFLKRFAEALPSDEEGESYSKEVPE MEKRYGGF MRF Fragments of Pro-Enkephalin, that may be determined in a bodily fluid, may be e.g.
  • SEQ ID NO.2 (Synenkephalin, Pro-Enkephalin 1-73) ECSQDCATCSYRLVRPADINFLACVMECEGKLPSLKIWETCKELLQLSKPELPQDGTSTL RENSKPEESHLLA SEQ ID NO.3 (Met-Enkephalin) YGGFM SEQ ID NO.4 (Leu-Enkephalin) YGGFL SEQ ID NO.5 (Pro-Enkephalin 90-109)
  • MDELYPMEPEEEANGSEILA SEQ ID NO 6: (Pro-Enkephalin 119-159, Mid-regional Pro-Enkephalin-fragment, MR-PENK) DAEEDDSLANSSDLLKELLETGDNRERSHHQDGSDNEEEVS SEQ ID NO.7 (Met-Enkephalin-Arg-Gly-Leu) YGGFMRGL SEQ ID NO.8 (Pro-Enkephalin 172-183) SPQLEDEAKELQ SEQ ID NO.9
  • Determining the level of Pro-Enkephalin including Leu-Enkephalin and Met-Enkephalin or fragments thereof may mean that the immunoreactivity towards Pro-Enkephalin or fragments thereof including Leu-Enkephalin and Met-Enkephalin is determined.
  • a binder used for determination of Pro-Enkephalin including Leu-Enkephalin and Met-Enkephalin or fragments thereof depending on the region of binding may bind to more than one of the above displayed molecules. This is clear to a person skilled in the art.
  • a binder is used in the methods of the present invention that binds to a region within the amino acid sequence of Pro-Enkephalin (PENK) in a bodily fluid
  • the terms “determining the level of Pro-Enkephalin (PENK) or fragments thereof in a bodily fluid obtained from said patient” are equivalent to “determining the level of immunoreactive analyte by using at least one binder that binds to a region within the amino acid sequence of Pro-Enkephalin (PENK) in a bodily fluid obtained from said patient”.
  • a binder is used in the methods of the present invention that binds to a region within the amino acid sequence of Pro- Enkephalin (PENK) in a bodily fluid.
  • said binder used in the methods of the present invention does bind to a region within the amino acid sequence of leu-enkephalin or met-enkephalin in a bodily fluid.
  • said at least one binder binds to mid-regional Pro-Enkephalin (MR-PENK) or a fragment thereof.
  • MR-PENK mid-regional Pro-Enkephalin
  • the level of immunoreactive analyte by using at least one binder that binds to a region within the amino acid sequence of any of the above peptides and peptide fragments, (i.e.
  • Pro-Enkephalin and fragments according to any of the sequences 1 to 12), is determined in a bodily fluid obtained from said subject; and correlated to the specific embodiments of clinical relevance.
  • the level of MR- PENK is determined (SEQ ID NO. 6: Pro-Enkephalin 119-159, Mid-regional Pro-Enkephalin- fragment, MR-PENK).
  • the level of immunoreactive analyte by using at least one binder that binds to MR-PENK is determined and is correlated to the above- mentioned embodiments according to the invention.
  • Level of immunoreactivity means the concentration of an analyte determined quantitatively, semi-quantitatively or qualitatively by a binding reaction of a binder to such analyte, where preferably the binder has an affinity constant for binding to the analyte of at least 108 M-1, and the binder may be an antibody or an antibody fragment or a non-Ig scaffold, and the binding reaction is an immunoassay.
  • the level of Proenkephalin or fragments thereof is determined by using at least one binder that binds to a region within the amino acid sequence of a peptide selected from the group comprising Pro-Enkephalin or fragments thereof of at least 5 amino acids.
  • said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • said binder do not bind to enkephalin peptides Met-Enkephalin SEQ ID No: 3, and Leu-Enkephalin SEQ ID No: 4.
  • said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No. 1, 2, 5, 6, 8, 9, 10 and 11. In another specific embodiment said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No.1, 2, 5, 6, 8 and 9. In another very specific embodiment said binder binds to Pro-Enkephalin 119-159, Mid-regional Pro-Enkephalin-fragment, MR-PENK (SEQ ID No.6). The before mentioned binder binds to said peptides in a bodily fluid obtained from said subject.
  • subject matter of the present invention is method for early prediction of a risk or monitoring of a risk or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Proenkephalin or fragments thereof by using at least one binder that binds to a region within the amino acid sequence of a peptide selected from the group comprising the Pro-Enkephalin peptides and fragments of SEQ ID No.1 to 12 in a bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof with the risk of and/ or severity of reduced graft function, wherein said reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • said binder is selected from the group comprising an antibody, an antibody fragment or a non-Ig-Scaffold binding to Pro-Enkephalin or fragments thereof of at least 5 amino acids.
  • the level of Pro-Enkephalin or fragments thereof are measured with an immunoassay using antibodies or fragments of antibodies binding to Pro-Enkephalin or fragments thereof.
  • An immunoassay that may be useful for determining the level of Pro-Enkephalin or fragments thereof of at least 5 amino acids may comprise the steps as outlined in Example 1. All thresholds and values have to be seen in correlation to the test and the calibration used according to Example 1. A person skilled in the art may know that the absolute value of a threshold might be influenced by the calibration used.
  • Another possibility is to determine with the assay in question, given this test has sufficient analytical sensitivity, the median biomarker level of a representative normal population, compare results with the median biomarker levels as described in the literature (e.g. Donato et al. 2018. Clin Biochem. 58: 72-77) and recalculate the calibration based on the difference obtained by this comparison.
  • said predetermined threshold level of Pro- Enkephalin or fragments thereof is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • said predetermined threshold level of Pro-Enkephalin or fragments thereof is an x-fold of the median of the level of Proenkephalin or fragments thereof in a healthy population.
  • the threshold level of Pro- Enkephalin or fragments thereof is in the range between the 1.0-fold and 15.6-fold, more preferred in the range between the 1.7-fold and 10.4-fold, even more preferred in the range between 2.1-fold and 8.3-fold, most preferred in the range between 3.1-fold and 6.2-fold of the median of the level of Proenkephalin or fragments thereof in a healthy population.
  • a prerequisite of using the x-fold of e.g. the median (or a specific percentile) of the level of Proenkephalin or fragments thereof in a healthy population as a threshold level is that the assay used for the present invention and the differently calibrated assay (as mentioned above) are measuring in a linear way.
  • the level of any of the above analytes may be determined by other analytical methods e.g. mass spectrometry.
  • Mass spectrometric (MS) methods may include matrix assisted laser desorption/ ionization MS (MALDI-MS), liquid chromatography-mass spectrometry (LC- MS) and liquid-chromatography electrospray ionization MS (LC-ESI-MS).
  • MALDI-MS matrix assisted laser desorption/ ionization MS
  • LC- MS liquid chromatography-mass spectrometry
  • LC-ESI-MS liquid-chromatography electrospray ionization MS
  • the binder to Pro-Enkephalin is selected from the group consisting of antibodies e.g. IgG, a typical full-length immunoglobulin, or antibody fragments containing at least the F-variable domain of heavy and/or light chain as e.g.
  • fragment antigen binding including but not limited to Fab-fragments including Fab minibodies, single chain Fab antibody, monovalent Fab antibody with epitope tags, e.g. Fab-V5Sx2; bivalent Fab (mini-antibody) dimerized with the CH 3 domain; bivalent Fab or multivalent Fab, e.g. formed via multimerization with the aid of a heterologous domain, e.g. via dimerization of dHLX domains, e.g.
  • Fab-dHLX-FSx2 F(ab‘)2-fragments, scFv-fragments, multimerized multivalent or/and multi- specific scFv-fragments, bivalent and/or bispecific diabodies, BITE® (bispecific T-cell engager), trifunctional antibodies, polyvalent antibodies, e.g. from a different class than G; single-domain antibodies, e.g. nanobodies derived from camelid or fish immunoglobulines.
  • the level of Pro-Enkephalin or fragments thereof are measured with an assay using binders selected from the group comprising aptamers, non-Ig scaffolds as described in greater detail below binding to Pro-Enkephalin or fragments thereof.
  • Binder that may be used for determining the level of Pro-Enkephalin or fragments thereof exhibit an affinity constant to Pro-Enkephalin of at least 107 M-1, preferred 108 M-1, preferred affinity constant is greater than 109 M-1, most preferred greater than 1010 M-1.
  • Binding affinity may be determined using the Biacore method, offered as service analysis e.g. at Biaffin, Kassel, Germany (http://www.biaffin.com/de/).
  • Biacore method offered as service analysis e.g. at Biaffin, Kassel, Germany (http://www.biaffin.com/de/).
  • biopolymer scaffolds are well known in the art to complex a target molecule and have been used for the generation of highly target specific biopolymers.
  • Non-Ig scaffolds may be protein scaffolds and may be used as antibody mimics as they are capable to bind to ligands or antigens.
  • Non-Ig scaffolds may be selected from the group comprising tetranectin-based non-Ig scaffolds (e.g. described in US 2010/0028995), fibronectin scaffolds (e.g. described in EP 1266025; lipocalin- based scaffolds (e.g. described in WO 2011/154420); ubiquitin scaffolds (e.g. described in WO 2011/073214), transferring scaffolds (e.g. described in US 2004/0023334), protein A scaffolds (e.g.
  • the threshold level is a level, which allows for allocating the patient into a group of patients who have been diagnosed and/ or are being at risk of an adverse event (e.g., reduced graft function), or into a group of patients who have not been diagnosed and/ or are not being at risk of an adverse event, or into a severity group (e.g., of delayed graft function).
  • the threshold level shall allow for differentiating between a patient who is diagnosed and/ or is at risk of an adverse event and a patient who is not diagnosed and/ or at risk of an adverse event. It is known in the art how threshold levels can be determined. Threshold levels are predetermined values and are set to meet routine requirements in terms of, e.g., specificity and/or sensitivity. These requirements can vary.
  • sensitivity or specificity has to be set to certain limits, e.g., 80%, 90%, 95% or 98%, respectively.
  • the sensitivity and specificity of a diagnostic and/or prognostic test depends on more than just the analytical "quality” of the test, they also depend on the definition of what constitutes an abnormal result.
  • Receiver Operating Characteristic curves are typically calculated by plotting the value of a variable versus its relative frequency in "reference group” (i.e. patients who do not develop reduced graft function after kidney transplantation) and "disease” populations (i.e. patients developing reduced graft function after kidney transplantation).
  • a distribution of marker levels for patients with and without developing reduced graft function will likely overlap.
  • a test does not absolutely distinguish patients with and without developing reduced graft function (e.g., slow graft function or delayed graft function) with 100% accuracy, and the area of overlap indicates where the test cannot distinguish normal from disease.
  • a threshold is selected, above which (or below which, depending on how a marker changes with the disease) the test is considered to be abnormal and below which the test is considered to be normal.
  • the area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results do not necessarily give an accurate number. As long as one can rank results, one can create a ROC curve.
  • a reference group may be a healthy population, e.g., with no signs and symptoms of a disease.
  • a reference group may be a population of kidney transplantation patients, in particular without developing reduced graft function (e.g., patients with immediate graft function).
  • a reference group may consist of more than one reference subjects.
  • the horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives.
  • the vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off threshold selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained.
  • the area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition.
  • the area under the ROC curve can be used to determine the effectiveness of the test.
  • threshold levels can further be obtained for instance from a Kaplan-Meier analysis, where the occurrence of a disease is correlated with e.g. the tertiles, quartiles, quintiles of the markers (e.g. kidney markers, cardiovascular markers) in the population.
  • markers e.g. kidney markers, cardiovascular markers
  • threshold values are for instance the 90th, 95th or 99th percentile of a normal population.
  • a higher percentile than the 75th percentile one reduces the number of false positive subjects identified, but one might miss to identify subjects, who are at moderate, albeit still increased risk.
  • the threshold value depending on whether it is considered more appropriate to identify most of the subjects at risk at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly the subjects at high risk at the expense of missing several subjects at moderate risk.
  • the 75th percentile, more preferred the 90th percentile, even more preferred a 95th percentile, most preferred the 99th percentile values can be used for the upper limits of the normal range.
  • thresholds for a specific indication may be determined using other methods. Such methods include eg the Youden optimum, thresholds that maximise overall accuracy, the odds ratio, or the positive or negative predictive value. In some situations, thresholds achieving a pre-specified level of sensitivity or specificity (eg 80%, 90%, 95% or 99%) can be appropriate for the clinical application. The choice of methods depends on the clinical application, which weights the costs of false positive and false negative results based on the test result consequences for the patient and the health care system, as well as the clinical need. Finally, multiple approaches may be combined to define a consensus threshold.
  • the threshold level may vary depending on various physiological parameters such as age, gender or sub-population, as well as on the means used for the determination of Pro-Enkephalin and fragments thereof referred to herein. In a specific embodiment of the invention, said threshold levels are age-dependent.
  • the values for MR-PENK (Proenkephalin 119-159; SEQ ID N: 6) revealed the use of more than one threshold value depending on the age of the subject. The threshold values decreased with increasing age of the patients.
  • said predetermined threshold level of Pro-Enkephalin or fragments thereof is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • said fragment of Pro-Enkephalin is MR-PENK (PENK 119-159, SEQ ID NO.6).
  • said predetermined threshold level of MR-PENK is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • Said threshold levels may be similar for other Pro-Enkephalin fragments that are derived from the precursor Proenkephalin in a 1:1 molar ratio.
  • said threshold for Met-Enkephalin is six-times higher compared to the threshold level of MR-PENK.
  • said predetermined threshold level of Proenkephalin or fragments thereof, in particular MR-PENK is 300 pmol/L if measured up to 24 hours after kidney transplantation.
  • said predetermined threshold level of Proenkephalin or fragments thereof in particular MR-PENK (PENK 119-159, SEQ ID NO.6) is 200 pmol/L if measured between more than 24 hours and up to 48 hours after kidney transplantation.
  • the level of Pro-Enkephalin or fragments thereof is determined in a sample of bodily fluid obtained from said patient before and after kidney transplantation. Said sample, which is obtained after kidney transplantation, is taken in the range between 3 and 96 hours, more preferred in the range between 6 and 72 hours, even more preferred in the range between 9 and 48 hours, most preferred in the range between 12 and 24 hours after kidney transplantation.
  • said sample which is obtained after kidney transplantation, is taken within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • the level of Pro-Enkephalin or fragments thereof may be determined more than 96 hours after kidney transplantation as follow-up.
  • a relative change of the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid taken (i) at least once before and at least once or (ii) at least twice after kidney transplantation is calculated and correlated with the diagnosis and/ or the risk of and/ or the severity of reduced graft function of the kidney of said patient.
  • said relative change of the level of Pro-Enkephalin or fragments thereof is an insufficient decrease or even an increase.
  • a reduced graft function of the kidney in said patient is diagnosed and/ or predicted if said decrease is less than 35%, preferably less than 40%, even more preferred less than 45%, most preferred less than 50% when the level of the earlier sample is set to 100%.
  • reduced graft function is diagnosed and/ or a risk of reduced graft function is predicted in said patient if said relative change between the level of Pro- Enkephalin or fragments thereof in the samples either taken (i) at least once before and at least once after or (ii) at least twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%.
  • sample 1 samples are taken for example once before (sample 1) and once (e.g., 24 hours) after kidney transplantation (sample 2) and a relative change of the level of Pro-Enkephalin or fragments thereof is calculated between sample 1, which is set 100% and sample 2, resulting in a decrease of less than 50% or an increase, said patient is diagnosed and/ or predicted to be at risk of reduced graft function.
  • a third sample may be taken after sample 2 (e.g., 48 hours) after kidney transplantation for monitoring of the patient.
  • a relative change of the level of Pro- Enkephalin or fragments thereof is calculated between sample 2, which is now set 100% and sample 3.
  • Subject matter of the invention is further a medicament for use in early treatment of reduced graft function in a kidney transplantation patient, wherein said patient is selected by a diagnostic method comprising the steps: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis of reduced graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • said medicament is selected from the group comprising recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, C1 esterase inhibitor.
  • the level of Pro Enkephalin or fragments thereof is measured with an immunoassay and said binder is an antibody, or an antibody fragment binding to Pro-Enkephalin or fragments thereof.
  • the assay used comprises two binders that bind to two different regions within the region of Pro-Enkephalin that is amino acid 133-140 (LKELLETG, SEQ ID No. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No.14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • the assay sensitivity for determining Pro-Enkephalin or fragments in a sample is ⁇ 15 pmol/L, preferably ⁇ 10 pmol/L and most preferred ⁇ 6 pmol/L.
  • Subject matter of the present invention is the use of at least one binder that binds to a region within the amino acid sequence of a peptide selected from the group comprising the peptides and fragments of SEQ ID No.1 to 12 in a bodily fluid obtained from said subject in a method for early prediction of a risk or monitoring of a risk or prediction of the severity of reduced graft function in a kidney transplantation patient.
  • said binder is selected from the group comprising an antibody, an antibody fragment or a non-Ig scaffold binding to Pro-Enkephalin or fragments thereof of at least 5 amino acids.
  • said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
  • said binder binds to enkephalin peptides met-enkephalin (SEQ ID No: 3), and leu-enkephalin (SEQ ID No: 4).
  • said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No. 1, 2, 5, 6, 8, 9, 10 and 11.
  • said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No. 1, 2, 5, 6, 8 and 9.
  • said binder binds to Pro-Enkephalin 119-159, mid-regional Pro-Enkephalin- fragment, MR-PENK (SEQ ID No.6).
  • the at least one binder binds to a region within the amino acid sequence of Pro-Enkephalin 119-159, mid-regional Pro-Enkephalin fragment, MR-PENK (SEQ ID No. 6) in a bodily fluid obtained from said subject, more specifically to amino acid 133-140 (LKELLETG, SEQ ID No.
  • At least one clinical parameter that may be determined is selected from the group comprising: alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl transpeptidase, calprotectin, C-C motif chemokine ligand 14, chitinase 3-like protein 1, hepatocyte growth factor, hepcidin, IL-18, beta-trace protein (BTP), cystatin C, KIM-1, TIMP-2, IGFBP-7, blood urea nitrogen (BUN), NGAL, liver-type fatty acid binding protein, monocyte chemoattractant peptide- 1, Creatinine Clearance, serum Creatinine (SCr), urea, metrin-1, osteopontin, retinol binding protein, tumor necrosis factor, and Apache Score.
  • alanine aminopeptidase alkaline phosphatase, gamma-glutamyl transpeptidase, calprotectin, C-C motif chemokine
  • said method is performed more than once in order to monitor the risk of said patient or in order to monitor the course of treatment of said kidney transplantation patient. In one specific embodiment said monitoring is performed in order to evaluate the response of said patient to preventive and/or therapeutic measures taken. In one embodiment of the invention the method is used in order to stratify said patients into risk groups. Said patient may be stratified into a risk group of low, intermediate or high risk of reduced graft function. Said patient may be stratified into a low, intermediate or high-risk group for slow graft function and/ or delayed graft function.
  • Subject matter of the invention is further an assay for determining Pro-Enkephalin and Pro- Enkephalin fragments in a sample comprising two binders that bind to two different regions within the region of Pro-Enkephalin that is amino acid 133-140 (LKELLETG, SEQ ID NO.13) and amino acid 152-159 (SDNEEEVS, SEQ ID NO.14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • it may be a so-called POC-test (point –of-care), that is a test technology which allows performing the test within less than 1 hour near the patient without the requirement of a fully automated assay system.
  • POC-test point –of-care
  • such an assay is a sandwich immunoassay using any kind of detection technology including but not restricted to enzyme label, chemiluminescence label, electrochemiluminescence label, preferably a fully automated assay.
  • such an assay is an enzyme labeled sandwich assay. Examples of automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®.
  • immunoassays are known and may be used for the assays and methods of the present invention, these include: radioimmunoassays ("RIA”), homogeneous enzyme-multiplied immunoassays (“EMIT”), enzyme linked immunoadsorbent assays (“ELISA”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays and immuno-chromatography assays.
  • RIA radioimmunoassays
  • EMIT homogeneous enzyme-multiplied immunoassays
  • ELISA enzyme linked immunoadsorbent assays
  • ARIS apoenzyme reactivation immunoassay
  • dipstick immunoassays dipstick immunoassays
  • immuno-chromatography assays immuno-chromatography assays.
  • at least one of said two binders is labeled in order to be detected.
  • the preferred detection methods comprise immunoassays in various formats such as for instance radioimmunoassay (RIA), chemiluminescence- and fluorescence-immunoassays, Enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, and rapid test formats such as for instance immunochromatographic strip tests.
  • said label is selected from the group comprising chemiluminescent label, enzyme label, fluorescence label, radioiodine label.
  • the assays can be homogenous or heterogeneous assays, competitive and non-competitive assays.
  • the assay is in the form of a sandwich assay, which is a non-competitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and to a second antibody.
  • the first antibody may be bound to a solid phase, e.g. a bead, a surface of a well or other container, a chip or a strip
  • the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety.
  • the amount of labeled antibody bound to the analyte is then measured by an appropriate method.
  • the assay comprises two capture molecules, preferably antibodies which are both present as dispersions in a liquid reaction mixture, wherein a first labelling component is attached to the first capture molecule, wherein said first labelling component is part of a labelling system based on fluorescence- or chemiluminescence-quenching or amplification, and a second labelling component of said marking system is attached to the second capture molecule, so that upon binding of both capture molecules to the analyte a measurable signal is generated that allows for the detection of the formed sandwich complexes in the solution comprising the sample.
  • chemiluminescence based assays comprise the use of dyes, based on the physical principles described for chemiluminescent materials in (Kirk-Othmer, Encyclopedia of chemical technology, 4th ed., executive editor, J. I. Kroschwitz; editor, M. Howe- Grant, John Wiley & Sons, 1993, vol.15, p.518-562, incorporated herein by reference, including citations on pages 551-562).
  • Chemiluminescent label may be acridinium ester label, steroid labels involving isoluminol labels and the like.
  • Preferred chemiluminescent dyes are acridiniumesters.
  • an “assay” or “diagnostic assay” can be of any type applied in the field of diagnostics. Such an assay may be based on the binding of an analyte to be detected to one or more capture probes with a certain affinity. Concerning the interaction between capture molecules and target molecules or molecules of interest, the affinity constant is preferably greater than 108 M-1.
  • “binder molecules” are molecules which may be used to bind target molecules or molecules of interest, i.e. analytes (i.e. in the context of the present invention PENK and fragments thereof), from a sample.
  • Binder molecules must thus be shaped adequately, both spatially and in terms of surface features, such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and/or acceptors, to specifically bind the target molecules or molecules of interest.
  • the binding may for instance be mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a combination of two or more of the aforementioned interactions between the capture molecules and the target molecules or molecules of interest.
  • binder molecules may for instance be selected from the group comprising a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, an antibody, a peptide or a glycoprotein.
  • the binder molecules are antibodies, including fragments thereof with sufficient affinity to a target or molecule of interest, and including recombinant antibodies or recombinant antibody fragments, as well as chemically and/or biochemically modified derivatives of said antibodies or fragments derived from the variant chain with a length of at least 12 amino acids thereof.
  • Chemiluminescent label may be acridinium ester label, steroid labels involving isoluminol labels and the like.
  • Enzyme labels may be lactate dehydrogenase (LDH), creatine kinase (CPK), alkaline phosphatase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), acidic phosphatase, glucose- 6-phosphate dehydrogenase, horse radish peroxidase (HRP) and so on.
  • LDH lactate dehydrogenase
  • CPK creatine kinase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • acidic phosphatase glucose- 6-phosphate dehydrogenase
  • HRP horse radish peroxidase
  • assays for determining Pro-Enkephalin or Pro-Enkephalin fragments in a sample according to the present invention such assay is a sandwich assay, preferably a fully automated assay.
  • ELISA fully automated or manual. It may be a so-called POC-test (point-of-care).
  • automated or fully automated assay comprise assays that may be used for one of the following systems: Roche Elecsys®, Abbott Architect®, Siemens Centauer®, Brahms Kryptor®, Biomerieux Vidas®, Alere Triage®, Ortho Vitros®. Examples of test formats are provided above.
  • at least one of said two binders is labeled in order to be detected. Examples of labels are provided above.
  • At least one of said two binders is bound to a solid phase.
  • solid phases are provided above.
  • said label is selected from the group comprising chemiluminescent label, enzyme label, fluorescence label, radioiodine label.
  • a further subject of the present invention is a kit comprising an assay according to the present invention wherein the components of said assay may be comprised in one or more container.
  • subject matter of the present invention is a point-of-care device for performing a method according to the invention, wherein said point-of-care device comprises at least one antibody or antibody fragment directed to either amino acid 133-140 (LKELLETG, SEQ ID No. 13) or amino acid 152-159 (SDNEEEVS, SEQ ID NO.14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • said point-of-care device comprises at least one antibody or antibody fragment directed to either amino acid 133-140 (LKELLETG, SEQ ID No. 13) or amino acid 152-159 (SDNEEEVS, SEQ ID NO.14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • subject matter of the present invention is a kit or performing a method according to the invention, wherein said point-of-care device comprises at least one antibody or antibody fragment directed to either amino acid 133-140 (LKELLETG, SEQ ID No.13) or amino acid 152-159 (SDNEEEVS, SEQ ID No.14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • subject matter of the present invention is a kit for performing a method according to the invention, wherein said point-of-care device comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No.13) and amino acid 152-159 (SDNEEEVS, SEQ ID No. 14), wherein each of said regions comprises at least 4 or 5 amino acids.
  • antibody generally comprises monoclonal and polyclonal antibodies and binding fragments thereof, in particular Fc-fragments as well as so called “single-chain-antibodies” (Bird et al. 1988), chimeric, humanized, in particular CDR-grafted antibodies, and dia- or tetrabodies (Holliger et al.1993). Also comprised are immunoglobulin-like proteins that are selected through techniques including, for example, phage display to specifically bind to the molecule of interest contained in a sample. In this context the term “specific binding” refers to antibodies raised against the molecule of interest or a fragment thereof.
  • An antibody is considered to be specific, if its affinity towards the molecule of interest or the aforementioned fragment thereof is at least preferably 50-fold higher, more preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to make antibodies and to select antibodies with a given specificity.
  • An antibody or fragment according to the present invention is a protein including one or more polypeptides substantially encoded by immunoglobulin genes that specifically binds an antigen.
  • Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length.
  • Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length.
  • Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH-terminus.
  • Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.
  • the basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions.
  • Immunoglobulins also exist in a variety of other forms inc1uding, for example, Fv, Fab, and (Fab')2, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al. 1987. Eur. J. Immunol.17: 105; Huston et al.1988. Proc.
  • An immune complex is an antibody, such as a monoclonal antibody, chimeric antibody, humanized antibody or human antibody, or functional antibody fragment, specifically bound to the antigen.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species.
  • the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
  • a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody, although other mammalian species can be used, or the variable region can be produced by molecular techniques.
  • Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDR’s.
  • the acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoc1onal antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions.
  • exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (e.g., see U.S. Patent No. 5,585,089).
  • a human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest.
  • Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell.
  • Human antibodies can also be produced by phage display methods (see, e.g., WO91/17271; WO92/001047; WO92/20791) or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (for example, see WO93/12227; WO 91/10741).
  • Humanization of murine antibodies may be conducted according to the following procedure: For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modelling (Almagro and Fransson 2008. Humanization of antibodies. Front Biosci. 13:1619-33).
  • the term monoclonal antibody is meant to include monoclonal antibodies, as well as fragments of monoclonal antibodies, such as the ones detailed herein, more particularly monoclonal antibodies.
  • Hybridoma in a further aspect, is a monoclonal antibody obtainable by a method comprising: i) fusing antibody-secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody.
  • the antibody according to the present invention is a monoclonal antibody obtainable by isolating from a multitude of hybridomas a hybridoma producing a desired monoclonal antibody, wherein said multitude of hybridomas were produced by fusing antibody- secreting cells from an animal previously immunized with an antigen with myeloma cells to obtain multitude of hybridomas.
  • a desired monoclonal antibody is in particular a monoclonal antibody binding the antigen, in particular with a binding affinity of at least 107 M-1, preferred 108 M-1, more preferred affinity is greater than 109 M-1, most preferred greater than 1010 M-1.
  • the myeloma cells are of the cell line SP2/0.
  • said fusing in step i) comprises PEG-assisted fusion, Sendai virus-assisted fusion or electric current-assisted fusion.
  • said isolating in step ii) comprises performing an antibody capture assay, an antigen capture assay, and/or a functional screen.
  • step ii) isolating the hybridoma producing a desired monoclonal antibody may involve cloning and re-cloning the hybridomas using the limiting-dilution technique.
  • said antigen capture assay comprises: a) binding the produced antibodies to a substrate, particularly a solid substrate, b) allowing antigen to bind to said antibodies, c) removing unbound antigen by washing, d) detecting bound antigen; or said antigen capture assay comprises: a) allowing an antigen to bind the produced antibodies to form an antibody-antigen complex, b) binding said antibody-antigen complex to a substrate, particularly a solid substrate, c) removing unbound antigen by washing, d) detecting bound antigen.
  • said isolating of step ii) comprises performing an enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, cell staining, immunoprecipitation, and/or a western blot.
  • said detecting of the antibody or the antigen is accomplished with an immunoassay.
  • the animal is a transgenic animal, in particular a transgenic mouse (wherein in particular the mouse immunoglobulin (Ig) gene loci have been replaced with human loci within the transgenic animal genome), such as HuMabMouse or XenoMouse.
  • the antigen comprises a peptide as described herein in Table 1, which in certain embodiments (in particular for immunization) may be conjugated to a protein, particularly a serum protein, more particularly a serum albumin, more particularly BSA.
  • the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) fusing splenocytes cells from a Balb/c mouse previously immunized with a peptide as described herein in Table 1 with SP2/0 myeloma cells using polyethylene glycol, to obtain a multitude of hybridomas, ii) isolating from said multitude of hybridomas a hybridoma producing a desired monoclonal antibody; more preferably, the method comprises: 1) growing hybridomas for a first period (in particular 2 weeks) in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement] 2) followed replacing HAT medium with HT Medium
  • an antibody having affinity to an antigen is in particular an antibody with a binding affinity of at least 107 M-1, preferred 108 M-1, more preferred affinity is greater than 109 M-1, most preferred greater than 1010 M-1.
  • the antibody according to the present invention is a monoclonal antibody obtainable by isolating at least one antibody from a culture derived from at least one cell strain which expressed at least one antibody having affinity to an antigen from an antibody gene library.
  • the antigen comprises a peptide as described herein in Table 1, which in certain embodiments may be bound to a solid phase.
  • the antibody gene library is a naive antibody gene library, particularly a human naive antibody gene library, more particularly in said library the antibodies are presented via phage display, i.e. on phages comprising a nucleotide sequence encoding for such respective antibody; more particularly the library HAL 7, HAL 8, or HAL 9, more particularly a library comprising the human naive antibody gene libraries HAL7/8.
  • screening comprises the use of an antigen, particularly an antigen containing a tag, more particularly a biotin tag, linked thereto via two different spacers.
  • such panning strategy includes a mix of panning rounds with non-specifically bound antigen and antigen bound specifically via the tag, in the case of a biotin tag, bound to streptavidin. In this way, the background of non-specific binders may be minimized.
  • the antibody in certain embodiments of the method for obtaining an antibody, in step i), in embodiments wherein the library is a phage display library, the antibody is isolated by isolating a phage presenting said antibody (and comprising a nucleotide sequence encoding for the antibody).
  • step ii) said cell strain is generated via introduction of a nucleotide sequence encoding for the antibody
  • the library in step i) is a phage display library
  • the isolated phage from step i) may be used to produce a bacterial strain, e.g. an E. coli strain, expressing the antibody.
  • a bacterial strain e.g. an E. coli strain
  • antibody may be isolated from the supernatant of the culture.
  • the term “one antibody” in the expression “at least one antibody” in particular may include more than one antibody molecule of antibodies having the same amino acid sequence.
  • This understanding applies, mutatis mutandis, to the term “one cell strain”.
  • more than one antibody referring to a multitude of antibodies having distinct amino acid sequences, respectively
  • Such method may involve the selection of clones that are positive for binding to the antigen, e.g. via a binding assay, e.g.
  • the antibody according to the present invention is a monoclonal antibody obtainable by a method comprising: i) isolating at least one antibody having affinity to an antigen from an antibody gene library comprising the human naive antibody gene libraries HAL7/8, by eluting phages carrying said antibody from the library; ii) generating at least one E. coli cell strain expressing said at least one antibody; iii) isolating the at least one antibody from the supernantant a culture of the at least one E. coli cell strain obtained in step ii).
  • an antibody fragment according to the present invention is produced by a method in volving enzymatic digestion of an antibody.
  • this method produces e.g. Fab or F(ab)2 antibody fragments.
  • this method involves digestion with pepsin or papain, which are optionally immobilized on a surface.
  • antibodies may be humanized by CDR-grafting, in particular by a process involving the steps: - extracting RNA from hybridomas expressing an antibody of interest (e.g.
  • RNA obtained by a method as described herein); - amplifying said extracted RNA via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest, to obtain to obtain a DNA product; - further amplifying said DNA product via PCR, in particular using semi-nested primer sets specific for antibody variable regions; - determining the sequence of the DNA product; - aligning said sequence with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).
  • antibodies may be humanized by aligning the sequence of a DNA product that was obtained by amplifying RNA extracted from hybridomas expressing an antibody of interest via RT-PCR, in particular with primer sets specific for the heavy and light chains of the antibody of interest and further amplifying the DNA obtained therefrom via PCR, in particular using semi-nested primer sets specific for antibody variable regions, with homologous human framework sequences to determine a humanized sequence for the variable heavy chain and the variable light chain sequences (of the desired antibody).
  • antibodies may be humanized by - determining the complementary determining regions (CDR), which may be accomplished by analyzing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen; - translplanting said CDR sequences into a human framework region.
  • CDR complementary determining regions
  • antibodies may be humanized by transplanting CDR sequences, which may preferably have been determined by analyzing the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen, into a human framework region.
  • variations in the amino acid sequence of the CDRs or FRs may be introduced to maintain structural interactions with the antigen (which may otherwise be abolished by introducing the human FR sequences), for instance by a random approach using phage display libraries or via directed approach guided by molecular modelling.
  • the DNA sequences encoding for antibodies determined as detailed herein can be transferred by known genetic engineering techniques into cells and used for production of the antibody.
  • Producing antibodies in a further aspect, the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: - culturing a cell strain comprising a nucleotide sequence encoding for the antibody; - isolating the antibody from said culture.
  • the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a culture of a cell strain comprising a nucleotide sequence encoding for said antibody.
  • the cell strain is produced as described herein above and may comprise bacterial cells, such as gram-negative bacteria, e.g. E. coli, Proteus mirabilis, or Pseudomonas putidas, gram-positive bacteria, e.g.
  • Bacillus brevis Bacillus subtilis, Bacillus megaterium, Lactobacilli such as Lactobacillus zeae/casei or Lactobacillus paracasei, or Streptomyces, such as Streptomyces lividans; eucariotic cells such as yest, e.g. Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, or Yarrowia lipolytica; fugi, such as filamentous fungi, e.g. of the genus Trichoderma of Aspergillus, such as A.
  • niger e.g. subgenus A. awamori
  • Aspergillus oryzae Trichoderma reesei
  • Chrysosporium such as C. lucknowense
  • protozoae such as Leishmania, e.g. L. tarentolae
  • insect cells such as insect cells transfected a Baculovirus, e.g. AcNPV, such as insect cell lines from Spodoptera frugiperda, e.g. Sf-9 or Sf-21, Drosophila melanogaster, e.g. DS2, or Trichopulsia ni, e.g.
  • High Five cells BTI- TN-5B1-4
  • mammalian cells such as hamster, e.g. Chinese hamster ovary such as K1-, DukX B11-, DG44, Lec13, or BHK, mouse, e.g. mouse myeloma such as NS0, Homo sapiens, e.g. Per.C6, AGE1.HN, HEK293.
  • the cells may be hybridoma cells, e.g. as described herein.
  • culturing may take place in a static suspension culture, an agitated suspension culture, a membrane-based culture, a matrix-based culture or a high cell density bioreactor; a vessel for such culturing may be selected from the group comprising a T- flask, a roller culture, a spinner culture, a stirred tank bioreactor, an airlift bioreactor, a static membrane-based or matrix-based culture system, a suspension bioreactor, a fluidized bed bioreactor, a ceramic bioreactor, a perfusion system, a hollow fiber bioreactor.
  • the cells may be immobilized on a matrix.
  • a high cell density bioreactor is in particular a culture system capable of generating cell densities greater than 10 ⁇ 8 cells/ml.
  • the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by a method comprising: - generating a transgenic plant or animal comprising a nucleotide sequence encoding for the antibody; - isolating the antibody from said plant or animal or a secretion or product of said plant or animal.
  • the antibody according to the present invention is a monoclonal antibody obtainable by the methods described herein, produced by isolating the antibody from a transgenic plant or transgenic animal or a secretion or product of a transgenic plant or transgenic animal having a nucleotide sequence encoding for the antibody.
  • Said animal may e.g., be selected from a chicken, a mouse, a rat, a rabbit, a cow, a goat, a sheep, a pig; said secretion or product may e.g. be milk or an egg.
  • Said plant may e.g. be selected from tobacco (N. tabacum or N.
  • the antibodies can in certain embodiments be isolated by physicochemical fractionation, e.g. size exclusion chromatography, precipitation, e.g. using ammonium sulphate, ion exchange chromatography, immobilized metal chelate chromatography gel filtration, zone electrophoresis; based on their classification e.g.
  • the antibody is encoded by a nucleotide sequence where the nucleotide sequence is a reverse transcription of an amino acid sequence from an antibody produced by one of the processes described herein.
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis and/ or the risk and/ or the severity of reduced graft function, wherein reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • a method for patient stratification and/ or patient selection for early treatment of reduced graft function in a kidney transplantation patient comprising: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - stratifying and/ or selecting said patient for early treatment of reduced graft function in correlation of said level of Pro-Enkephalin or fragments thereof in said sample, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 or 2 or 5, wherein samples of bodily fluid are taken from said patient (i) at least once before and at least once after or (ii) at least twice after kidney transplantation and a relative change of the level of Pro-Enkephalin or fragments thereof is calculated, wherein the relative change correlates with the diagnosis and/ or the risk and/ or the severity of reduced graft function of the kidney. 7.
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 to 7, wherein, (i) if said at least one sample of bodily fluid is taken after kidney transplantation, said sample is obtained within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity reduced graft function in a kidney transplantation patient according to embodiments 1 to 9, wherein said patient is in need of renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive therapy and/ or adjustment of nephrotoxic drugs if reduced graft function is diagnosed and/ or a risk of reduced graft function is predicted in said patient.
  • immunosuppressive therapy selected from the group comprising interleukin 2 receptor antagonist, calcineurin inhibitors (cyclosporine A, tacrolimus), mammalian target of rapamycin inhibitor, corticosteroids, mycophenolate, sirolimus and azathioprine).
  • nephrotoxic drugs are selected from the group comprising calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), pain medications (e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin), anti-microbials (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin and vancomycin), cholesterol-lowering statins, angiotensin- converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g.
  • calcineurin inhibitors for immunosuppression e.g., cyclosporine A, tacrolimus
  • pain medications e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin
  • anti-microbials e.g., aminoglycoside
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient wherein additionally at least one clinical parameter is determined selected from the group comprising: alanine aminopeptidase, alkaline phosphatase, gamma-glutamyl transpeptidase, calprotectin, C-C motif chemokine ligand 14, chitinase 3-like protein 1, hepatocyte growth factor, hepcidin, IL-18, beta-trace protein (BTP), cystatin C, KIM-1, TIMP-2, IGFBP-7, blood urea nitrogen (BUN), NGAL, liver-type fatty acid binding protein, monocyte chemoattractant peptide-1, Creatinine Clearance, serum Creatinine (SCr), urea, metrin-1, osteopontin, retinol binding protein, tumor necrosis factor, and Apache Score.
  • a method for early diagnosis and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising determining the level of Pro- Enkephalin or fragments thereof in a sample of bodily fluid by using at least one binder, wherein said at least one binder binds to a region within the amino acid sequence selected from the group comprising SEQ ID No.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, preferably said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No.1, 2, 5, 6, 8, 9, 10 and 11, preferably said at least one binder binds to a region with the sequences selected from the group comprising SEQ ID No.1, 2, 5, 6, 8 and 9, preferably said at least one binder binds to SEQ ID No.6.
  • Method for early treatment of reduced graft function in a kidney transplantation patient wherein the treatment is selected from the group comprising renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive therapy and/ or adjustment of nephrotoxic drugs
  • said patient is selected by a diagnostic method comprising the steps: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis of reduced graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient wherein said patient is selected by a diagnostic method comprising the steps: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis of reduced graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • a point-of-care device for performing a method according to embodiments 1 to 27, wherein said point of care device comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No.14).
  • kit for performing a method according to embodiments 1 to 27, wherein said kit comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No.14).
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with graft function and/ or the risk and/ or the severity of reduced graft function, wherein reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 or 2 of aspect B, wherein if said level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient is elevated above a predetermined threshold level, said patient has reduced graft function and/ or is at risk of reduced graft function and/or is stratified and/ or selected for early treatment of reduced graft function. 4.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 to 4 of aspect B, wherein the level of Pro-Enkephalin or fragments thereof is determined in a sample of bodily fluid obtained from said patient (i) at least once before and at least once after or (ii) at least once after kidney transplantation.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 or 2 or 5 of aspect B, wherein samples of bodily fluid are taken from said patient (i) at least once before and at least once after or (ii) at least twice after kidney transplantation and a relative change of the level of Pro- Enkephalin or fragments thereof is calculated, wherein the relative change correlates with graft function and/ or the risk and/ or the severity of reduced graft function of the kidney. 7.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiment 6 of aspect B, wherein reduced graft function is diagnosed and/ or a risk of reduced graft function is predicted in said patient if said relative change between the level of Pro-Enkephalin or fragments thereof in the samples either taken (i) at least once before and at least once after or (ii) at least twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 to 7 of aspect B, wherein, (iv) if said at least one sample of bodily fluid is taken after kidney transplantation, said sample is obtained within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 to 8 of aspect B, wherein said determination of Pro-Enkephalin or fragments thereof is performed as follow-up measurement more than once after kidney transplantation in said patient.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity reduced graft function in a kidney transplantation patient according to embodiments 1 to 9 of aspect B, wherein said patient is in need of renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive therapy and/ or adjustment of nephrotoxic drugs if reduced graft function is diagnosed and/ or a risk of reduced graft function is predicted in said patient.
  • a method for early diagnosis of graft function and/ or early prediction of a risk and/ or monitoring of a risk and/ or prediction of the severity of reduced graft function in a kidney transplantation patient according to embodiments 1 to 12 of aspect B, wherein said patient is under immunosuppressive therapy selected from the group comprising interleukin 2 receptor antagonist, calcineurin inhibitors (cyclosporine A, tacrolimus), mammalian target of rapamycin inhibitor, corticosteroids, mycophenolate, sirolimus and azathioprine). 14.
  • immunosuppressive therapy selected from the group comprising interleukin 2 receptor antagonist, calcineurin inhibitors (cyclosporine A, tacrolimus), mammalian target of rapamycin inhibitor, corticosteroids, mycophenolate, sirolimus and azathioprine).
  • nephrotoxic drugs are selected from the group comprising calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), pain medications (e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin), anti-microbials (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g.
  • calcineurin inhibitors for immunosuppression e.g., cyclosporine A, tacrolimus
  • pain medications e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin
  • anti-microbials e.g., aminoglycosides
  • Method for early treatment of reduced graft function in a kidney transplantation patient wherein the treatment is selected from the group comprising renal replacement therapy and/ or administration of a medicament and/ or adjustment of immunosuppressive therapy and/ or adjustment of nephrotoxic drugs
  • said patient is selected by a diagnostic method comprising the steps: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - correlating said level of Pro-Enkephalin or fragments thereof in said sample with graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient wherein said patient is selected by a diagnostic method comprising the steps: - determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient and - correlating said level of Pro-Enkephalin or fragments thereof in said sample with the diagnosis of reduced graft function and/ or the risk of reduced graft function, wherein the reduced graft function is slow graft function (SGF) or delayed graft function (DGF) of the kidney.
  • SGF slow graft function
  • DGF delayed graft function
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiment 19 of aspect B wherein said medicament is selected from the group comprising recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, C1 esterase inhibitor.
  • said medicament is selected from the group comprising recombinant alkaline phosphatase, pegylated carboxyhemoglobin, relaxin, hepatocyte growth factor, mirocept, C1 esterase inhibitor.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiment 21 of aspect B wherein said predetermined threshold level is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • said predetermined threshold level is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • the level of Pro- Enkephalin or fragments thereof is determined in a sample of bodily fluid obtained from said patient (i) at least once before and at least once after or (ii) at least once after kidney transplantation. 24.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiments 19 to 23 of aspect B, wherein samples of bodily fluid are taken from said patient (i) at least once before and at least once after or (ii) at least twice after kidney transplantation and a relative change of the level of Pro-Enkephalin or fragments thereof is calculated, wherein the relative change correlates with graft function and/ or the risk and/ or the severity of reduced graft function of the kidney. 25.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiment 24 of aspect B, wherein reduced graft function is diagnosed and/ or a risk of reduced graft function is predicted in said patient if said relative change between the level of Pro-Enkephalin or fragments thereof in the samples either taken (i) at least once before and at least once after or (ii) at least twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient wherein (i) if said at least one sample of bodily fluid is taken after kidney transplantation, said sample is obtained within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation, or wherein (ii) if said at least two samples of bodily fluids are taken before and after kidney transplantation, said sample of bodily fluid taken after kidney transplantation is obtained, within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation, or wherein (iii) if said at least two samples of bodily fluids are taken after kidney transplantation, said first sample after kidney transplantation is obtained within 96 hours, more preferred within 72 hours, even more preferred within 48 hours, even more preferred within 24 hours, most preferred within 12 hours after kidney transplantation.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiment 28 of aspect B, wherein said renal replacement therapy is selected from the group comprising dialysis (hemodialysis or peritoneal dialysis), hemofiltration, and hemodiafiltration.
  • nephrotoxic drugs are selected from the group comprising calcineurin inhibitors for immunosuppression (e.g., cyclosporine A, tacrolimus), pain medications (e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin), anti-microbials (e.g., aminoglycosides, cephalosporins, penicillins, quinolones, rifampin and vancomycin), cholesterol-lowering statins, angiotensin-converting enzyme inhibitors (ACE inhibitors), angiotensin receptor blockers (ARBs), and diuretics, chemotherapeutic agents (e.g.
  • calcineurin inhibitors for immunosuppression e.g., cyclosporine A, tacrolimus
  • pain medications e.g., Nonsteroidal anti-inflammatory drugs (NSAIDs) as ibuprofen, aspirin
  • anti-microbials e.g., aminoglycosides
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiments 19 to 30 of aspect B, wherein said Pro-Enkephalin or fragment is selected from the group comprising SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No.4, SEQ ID No.5, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12.
  • Medicament for use in early treatment of reduced graft function in a kidney transplantation patient according to embodiments 19 to 31 of aspect B, wherein said sample of bodily fluid may be selected from the group comprising whole blood, serum, plasma, urine, cerebrospinal liquid (CSF), and saliva.
  • said sample of bodily fluid may be selected from the group comprising whole blood, blood serum, blood plasma. 34.
  • a point-of-care device for performing a method according to claims 1 to 16 of aspect B, wherein said point of care device comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No.13) and amino acid 152-159 (SDNEEEVS, SEQ ID No.14).
  • said kit comprises at least two antibodies or antibody fragments directed to amino acid 133-140 (LKELLETG, SEQ ID No. 13) and amino acid 152-159 (SDNEEEVS, SEQ ID No.14).
  • a method for prediction of kidney function in a kidney transplantation patient comprising: ⁇ determining the level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient; and ⁇ correlating said level of Pro-Enkephalin or fragments thereof in said sample with the kidney function of said patient, wherein said kidney function is defined as glomerular filtration rate (GFR), creatinine clearance rate (CCr), serum creatinine (SCr), urinalysis, blood urea nitrogen or urine output.
  • GFR glomerular filtration rate
  • CCr creatinine clearance rate
  • SCr serum creatinine
  • urinalysis blood urea nitrogen or urine output.
  • kidney function in a kidney transplantation patient wherein if said level of Pro-Enkephalin or fragments thereof in a sample of bodily fluid obtained from said patient is elevated above a predetermined threshold level a reduced kidney function is predicted.
  • said prediction is within 12 months, more preferred within 9 months, more preferred within 6 months, more preferred within 3 months, more preferred within 1 month, most preferred within 14 days.
  • the method for prediction of kidney function in a kidney transplantation patient according to embodiment 38 of aspect B wherein said prediction is a short-term prediction within 3 months, preferably within 1 month, more preferred within 28 days, even more preferred within 21 days, most preferred within 14 days.
  • said predetermined threshold level is in the range between 50 and 750 pmol/l, more preferred in the range between 100 and 500 pmol/l, even more preferred in the range between 150 and 400 pmol/l, most preferred in the range between 200 and 300 pmol/l.
  • the method for prediction of kidney function in a kidney transplantation patient according to embodiments 36 to 40 of aspect B wherein the level of Pro-Enkephalin or fragments thereof is determined in a sample of bodily fluid obtained from said patient (i) at least once before and at least once after or (ii) at least once after kidney transplantation.
  • the method for prediction of kidney function in a kidney transplantation patient according to embodiments 36 to 40 of aspect B wherein samples of bodily fluid are taken from said patient (i) at least once before and at least once after or (ii) at least twice after kidney transplantation and a relative change of the level of Pro-Enkephalin or fragments thereof is calculated, wherein the relative change correlates with said kidney function.
  • kidney function in a kidney transplantation patient according to embodiment 42 of aspect B, wherein a reduction in kidney function is predicted in said patient if said relative change between the level of Pro-Enkephalin or fragments thereof in the samples either taken (i) at least once before and at least once after or (ii) at least twice after kidney transplantation is either a decrease of less than 50% or an increase, when the level of the earlier sample is set to 100%.
  • the method for prediction of kidney function in a kidney transplantation patient according to embodiments 36 to 43 of aspect B wherein said Pro-Enkephalin or fragment is selected from the group comprising SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No. 11 and SEQ ID No.12.
  • said sample of bodily fluid may be selected from the group comprising whole blood, serum, plasma, urine, cerebrospinal liquid (CSF), and saliva. 46.
  • Peptides/ conjugates for Immunization Peptides for immunization (Table 1) were synthesized (JPT Technologies, Berlin, Germany) with an additional N-terminal Cysteine residue for conjugation of the peptides to bovine serum albumin (BSA). The peptides were covalently linked to BSA by using Sulfo-SMCC (Perbio Science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.
  • Table 1 immunization peptides and antibody names Peptide for immunization Pro-Enkephalin-sequence Antibody name (C)DAEEDD 119-125 NT-MR-PENK (C)EEDDSLANSSDLLK 121-134 NM-MR-PENK (C)LKELLETG 133-140 MR-MR-PENK (C)TGDNRERSHHQDGSDNE 139-155 MC-MR-PENK (C)SDNEEEVS 152-159 CT-MR-PENK
  • the antibodies were generated according to the following method: A BALB/c mouse was immunized with 100 ⁇ g peptide-BSA-conjugate at day 0 and 14 (emulsified in 100 ⁇ l complete Freund’s adjuvant) and 50 ⁇ g at day 21 and 28 (in 100 ⁇ l incomplete Freund’s adjuvant).
  • the animal received 50 ⁇ g of the conjugate dissolved in 100 ⁇ l saline, given as one intraperitoneal and one intravenous injection.
  • Splenocytes from the immunized mouse and cells of the myeloma cell line SP2/0 were fused with 1 ml 50 % polyethylene glycol for 30 s at 37 °C. After washing, the cells were seeded in 96-well cell culture plates.
  • Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20 % fetal calf serum and HAT-supplement]. After two weeks the HAT medium is replaced with HT Medium for three passages followed by returning to the normal cell culture medium.
  • the cell culture supernatants were primary screened for antigen specific IgG antibodies three weeks after fusion.
  • the positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and re-cloned using the limiting- dilution technique and the isotypes were determined. (Lane, R.D.1985 J. Immunol. Meth.81: 223-228; Ziegler, B. et al.1996. Horm. Metab. Res.28: 11-15).
  • Monoclonal antibody production Antibodies were produced via standard antibody production methods (Marx et al.1997. ATLA 25, 121) and purified via Protein A-chromatography.
  • the antibody purities were > 95 % based on SDS gel electrophoresis analysis. Labelling and coating of antibodies. All antibodies were labelled with acridinium ester according the following procedure: Labelled compound (tracer): 100 ⁇ g (100 ⁇ l) antibody (1 mg/ml in PBS, pH 7.4), was mixed with 10 ⁇ l Acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany) (EP 0353971) and incubated for 20 min at room temperature.
  • Labelled antibody was purified by gel-filtration HPLC on Bio-Sil SEC 400-5 (Bio-Rad Laboratories, Inc., USA) The purified labelled antibody was diluted in (300 mmol/l potassium phosphate, 100 mmol/l NaCl, 10 mmol/l Na-EDTA, 5 g/l bovine serum albumin, pH 7.0). The final concentration was approx. 800.000 relative light units (RLU) of labelled compound (approx. 20 ng labelled antibody) per 200 ⁇ l. Acridiniumester chemiluminescence was measured by using an AutoLumat LB 953 (Berthold Technologies GmbH & Co. KG).
  • Solid phase antibody coated antibody: Polystyrene tubes (Greiner Bio-One International AG, Austria) were coated (18 h at room temperature) with antibody (1.5 ⁇ g antibody/0.3 ml 100 mmol/l NaCl, 50 mmol/l Tris/HCl, pH 7.8). After blocking with 5 % bovine serum albumin, the tubes were washed with PBS, pH 7.4 and vacuum dried. Antibody specificity Antibody cross-reactivities were determined as follows: 1 ⁇ g peptide in 300 ⁇ l PBS, pH 7.4 was pipetted into Polystyrene tubes and incubated for 1h at room temperature. After incubation the tubes were washed 5 times (each 1ml) using 5% BSA in PBS, pH 7.4.
  • Each of the labelled antibodies were added (300 ⁇ l in PBS, pH 7.4, 800.000 RLU/ 300 ⁇ l) an incubated for 2h at room temperature, After washing 5 times (each 1ml of washing solution (20 mmol/l PBS, pH 7.4, 0.1 % Triton X 100), the remaining luminescence (labelled antibody) was quantified using the AutoLumat Luminometer 953. Synthetic MR-PENK peptide was used as reference substance (100%). The cross-reactivities of the different antibodies are listed in table 2.
  • Table 2 cross-reactivities of the different PENK-antibodies Antibody DAEE EEDDSLAN LKELLE TGDNRERSH SDNEEE MR-PENK DD SSDLLK TG HQDGSDNE VS (SEQ ID NO. 6) NT-MR- 121 10 ⁇ 1 ⁇ 1 ⁇ 1 100 PENK NM-MR- ⁇ 1 98 ⁇ 1 ⁇ 1 ⁇ 1 100 PENK MR-MR- ⁇ 1 ⁇ 1 105 ⁇ 1 ⁇ 1 100 PENK MC-MR- ⁇ 1 ⁇ 1 ⁇ 1 115 ⁇ 1 100 PENK CT-MR- ⁇ 1 ⁇ 1 ⁇ 1 ⁇ 1 95 100 PENK All antibodies bound the MR-PENK peptide, comparable to the peptides which were used for immunization.
  • Pro-Enkephalin Immunoassay 50 ⁇ l of sample (or calibrator) was pipetted into coated tubes, after adding labelled antibody (200ul), the tubes were incubated for 2 h at 18-25 °C. Unbound tracer was removed by washing 5 times (each 1 ml) with washing solution (20 mmol/l PBS, pH 7.4, 0.1 % Triton X-100). Tube- bound labelled antibody was measured by using the Luminometer 953. Using a fixed concentration of 1000pmol/ of MR-PENK.
  • the signal (RLU at 1000pmol MR-PENK/l) to noise (RLU without MR-PENK) ratio of different antibody combinations is given in table 3. All antibodies were able to generate a sandwich complex with any other antibody. Surprisingly, the strongest signal to noise ratio (best sensitivity) was generated by combining the MR-MR-PENK- and CT-MR-PENK antibody. Subsequently, we used this antibody combination to perform the MR-PENK- immunoassay for further investigations. MR-MR-PENK antibody was used as coated tube antibody and CT-MR-PENK antibody was used as labelled antibody.
  • Table 3 signal to noise ratio of different antibody combinations Solid NT-MR- NM-MR- MR-MR- MC-MR-PENK CT-MR- phase PENK PENK PENK PENK antibody Labelled antibody NT-MR- / 27 212 232 ⁇ 1 PENK NM-MR- 36 / 451 487 ⁇ 1 PENK MR-MR- 175 306 / 536 1050 PENK MC-MR- 329 577 542 / ⁇ 1 PENK CT-MR- ⁇ 1 615 1117 516 / PENK Calibration: The assay was calibrated, using dilutions of synthetic MR-PENK, diluted in 20 mM K2PO4, 6 mM EDTA, 0.5% BSA, 50 ⁇ M Amastatin, 100 ⁇ M Leupeptin, pH 8.0.
  • Figure 1 shows a typical Pro- Enkephalin dose / signal curve.
  • the assay sensitivity was 20 determinations of calibrator zero (no addition of MR-PENK) + 2SD) 5.5 pmol/L.
  • Example 2 The aim of this study was to evaluate the capabilities of the biomarker proenkephalin A 119-159 (PENK or penKid) to predict reduced graft function, specifically slow graft function (SGF) and delayed graft function (DGF) of the kidney in kidney transplant patients compared to serum creatinine (SCr). Sample collection and analysis EDTA-plasma samples were obtained from 159 patients.
  • PENK or penKid biomarker proenkephalin A 119-159
  • SGF slow graft function
  • DGF delayed graft function
  • Plasma samples were measured for SCr with a commercial enzymatic assay.
  • SGF Slow graft function
  • IGF immediate graft function
  • Statistical analysis Values are expressed as medians and interquartile ranges (IQR), or counts and percentages, as appropriate. Group comparisons of continuous variables were performed using Kruskal-Wallis test, with post-hoc tests for variables with more than two categories. Biomarker data (SCr and PENK) were log-transformed. Categorical data were compared using Pearson's Chi-squared Test for Count Data. Logistic regression was used to evaluate and compare PENK and SCr for their ability to predict DGF. To demonstrate independence from clinical variables, the added value of PENK on top of a multivariable model with the most relevant clinical variables to predict DGF was evaluated based on the likelihood ratio chi-square test for nested models.
  • IQR interquartile ranges
  • the concordance index (C index or AUC) is given as an effect measure for uni- and multivariable models. For multivariable models, a bootstrap corrected version of the C index/AUC is given. Receiver- operating-characteristic (ROC) curves were constructed and plotted to assess the sensitivity and specificity of PENK measurements obtained at various time points to predict and diagnose DGF. Change in PENK and SCr was computed as %-level of biomarker concentration prior to transplantation. All statistical tests were 2-tailed and a two-sided p-value of 0.05 was considered for significance.
  • ROC Receiver- operating-characteristic
  • Exemplary threshold values with the respective sensitivity and specificity are as follows: Using an absolute PENK cut-off at 300 pmol/L at day 1 after transplantation, patients with DGF can be identified with a sensitivity of 95% and specificity of 45% (odds ratio 16.2). Using an absolute PENK cut-off at 200 pmol/L at day 2 after transplantation, patients with DGF can be identified with a sensitivity of 97% and specificity of 40% (odds ratio 22.7). Using an absolute PENK cut-off at 200 pmol/L at day 3 post transplantation, patients with DGF can be identified with a sensitivity of 95% and specificity of 45% (odds ratio 16.2).
  • patients with DGF can be identified with a sensitivity of 89% and specificity of 41% (odds ratio 5.8).
  • patients with DGF can be identified with a sensitivity of 71% and specificity of 79% (odds ratio 9.0).
  • patients with DGF can be identified with a sensitivity of 65% and specificity of 93% (odds ratio 24.0).
  • logistic regression for the DGF endpoint Table 6
  • PENK is superior to SCr on day 1, 2 and 3 (all p ⁇ 0.0001) (Fig. 7 A).
  • Change in PENK is also superior to change in SCr for change at day 1, 2 and 3 (all p ⁇ 0.0001) (Fig.7 B).
  • the AUC for PENK increases from 0.81 on day 1 to 0.88 on day 2 and 0.91 on day 3.
  • change in PENK at day 1 already achieves a similar AUC (0.87), hinting at a gain in time if trajectories from pre-Tx are considered.
  • Table 7 shows that both, PENK at d1 and change in PENK at d1, provide added value on top of a multivariable model comprising of known clinical risk factors for DGF (both p ⁇ 0.0001).
  • PENK or change of PENK are in both cases the strongest predictor within the model (both Chi2 >15) and cold ischemia time being the second strongest factor in both models (both Chi2 >5).
  • Table 6 Logistic regression results for endpoint DGF for PENK, SCr, as well as pre-Tx change of PENK and SCr for the first 3 days post transplantation.
  • Model Chi2 Chi2 statistic; d.f.: degrees of freedom;
  • LR p-value likelihood ratio p-value;
  • C-Index concordance index or AUC; CI: confidence interval.
  • Figure 7 A (absolute) and 7 B (change) illustrate the differences in PENK and SCr for prediction of DGF for day 1, 2 and 3 post transplantation.
  • Table 7 Multivariable logistic regression results for endpoint DGF for a model comprising of donor age, donor SCr, living vs. deceased donor, cold ischemia time and patients’ duration on RRT pre-Tx (model ‘Multi’) and said model in combination with PENK at d1 (model ‘Multi, PENK’) or change in PENK at d1 (model ‘Multi, chng PENK’).
  • Model Chi2 Chi2 statistic; d.f.: degrees of freedom; LR p-value: likelihood ratio p-value; C-Index: bootstrap-corrected concordance index or AUC.
  • Fig.8 A and B show that PENK also discriminates between DGF severities earlier than SCr. While SCr in DGF severity 0-1 only fall after two weeks (day 12-15, post-hoc p-value 0.0010), this group declines in PENK already after week 1 (day 6-8, post hoc p-value for PENK ⁇ 0.0001 and for SCr 0.6113, comparing severity groups 0-1 with >7).
  • patients with DGF can be identified with a sensitivity of 95% and specificity of 57% (odds ratio 25.4).
  • PENK can discriminate patients with delayed graft function from patients with primary graft uptake earlier than SCr. In contrast to SCr, PENK was able to distinguish between DGF and no DGF as early as 24 hours after transplantation measured as single value and compared to a threshold or as a relative change.
  • PENK can discriminate between SGF and DGF and can predict duration of DGF (as reflected by DGF severity) much earlier than SCr.
  • SCr serum creatinine
  • eGFR estimated glomerular filtration rate
  • Ethylenediaminetetraacetic acid (EDTA) plasma samples were collected at the day of transplantation (0-12 hours prior to transplantation, day 0 or pre-Tx) and up to one day after transplantation (12-24 hours after transplantation, day 1). Serum creatinine measurement was performed via enzymatic methods on an automated chemical analyser (Konelab 20XT, Thermo Fisher Scientific, Waltham, MA, USA). For measurement of PENK a nonautomated immunoluminometric assay as described in Donato et al.2018 was used. (Donato et al.2018. Clin Biochem.58: 72-77).
  • Delayed graft function was defined as requirement of renal replacement therapy (RRT) in the first seven days after transplantation, differentiating DGF severity 0/1 (RRT only on day 0 or day 1, i.e. up to 24 hours after transplantation), DGF severity 2-7 (RRT ended between day 2 and day 7) and DGF severity >7 (RRT lasted longer than day 7).
  • Slow graft function SGF was defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 ⁇ 0.7.
  • Immediate graft function (IGF) was defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 > 0.7.
  • Reduced eGFR was defined as eGFR ⁇ 30 ml/min/1.73 m2.
  • the eGFR was determined via the latest CKD-EPI equation (2021). (Inker et al.2021. N Eng J Med.385:1737-1749).
  • Statistical analysis Medians and interquartile ranges (IQRs) or counts and percentages are reported, as appropriate.
  • IQRs interquartile ranges
  • the Kruskal-Wallis test was used for group comparisons of continuous variables, with post-hoc tests for variables with more than two categories. Categorical data were compared via Pearson's chi-square test for count data. All statistical tests were 2-tailed and a two-sided p-value of 0.05 was considered for significance.
  • PENK and SCr data were log-transformed and investigated via logistic regression for prediction of DGF.
  • Receiver operating characteristics (ROC) curves were constructed, and the area under the ROC curve (AUC) and the concordance index (C index) were calculated.
  • AUC area under the ROC curve
  • C index concordance index
  • R version 4.2.2 http://www.r-project.org, libraries rms, Hmisc, ROCR
  • IBM SPSS Statistics Version 22 SPSS Inc., Chicago, Illinois, USA
  • PENK was significantly increased in patients that developed DGF compared to patients without DGF (no DGF, including both, IGF and SGF) (573.5 [507.9-709.8] pmol/L vs.234.4 [163.6-361] pmol/L, p ⁇ 0.0001) (Table 2, Figure 10 A).
  • PENK distinguishes between patients that require RRT after transplantation (DGF) and patients that do not require RRT after transplantation (IGF and SGF) as early as 12-24 hours after transplantation allowing timely treatment decisions, while this cannot be achieved by the state-of-the-art biomarker SCr.
  • DGF RRT after transplantation
  • IGF and SGF RRT after transplantation
  • biomarker levels before transplantation did not differ significantly ( Figure 14).
  • delta PENK was able to predict of reduced eGFR at day 30 (AUC 0.82), while SCr was not useful (AUC 0.55), as displayed in the ROC curves ( Figure 15).
  • the C-index of delta PENK (0.82 [0.66- 0.99]) was superior to delta SCr (0.54 [0.31-0.79]), as was PENK on day 1 (0.74 [0.59-0.89]) compared to SCr on day 1 (0.53 [0.33-0.74]).
  • PENK can predict delayed graft function, differentiate between SGF and DGF, grade DGF severity and predict reduced eGFR at day 30 as early as 12-24 hours after kidney transplantation, while SCr measured on day 1 after transplantation is not able to identify these high-risk patients.
  • Fig 2A - Box and Whisker Plot of PENK by DGF at time points prior to transplantation (pre-Tx), on day 1, 2, 3, after one week (day 6-8: day 7, or day 6 or 8 if day 7 was missing), two weeks (day 12-15: day 14, or day 12, 13 or 15 if day 14 was missing) and three weeks (day 21/last: day 21 or last observed prior to discharge).
  • Fig.2B Box and Whisker Plot of SCr by DGF at time points prior to transplantation (pre-Tx), on day 1, 2, 3, after one week (day 6-8: day 7, or day 6 or 8 if day 7 was missing), two weeks (day 12-15: day 14, or day 12, 13 or 15 if day 14 was missing) and three weeks (day 21/last: day 21 or last observed prior to discharge). ). Cut-off line at 1.2 mg/dL.
  • Fig.3B Box and Whisker Plot of change of SCr (as percent from pre-Tx) in patients with DGF and non-DGF. Timepoints as defined in figure 5 A and B. Cut-off line at 50% from pre-Tx concentration.
  • Fig.4B Box and Whisker Plot of SCr by DGF, SGF and IGF at time points prior to transplantation (pre-Tx), on day 1, 2, 3, after one week (day 6-8: day 7, or day 6 or 8 if day 7 was missing), two weeks (day 12-15: day 14, or day 12, 13 or 15 if day 14 was missing) and three weeks (day 21/last: day 21 or last observed prior to discharge). Cut-off line at 1.2 mg/dL.
  • Fig.5A Box and Whisker Plot of change of PENK (as percent from pre-Tx) in patients with IGF, SGF and DGF. Timepoints as defined in figure 4 A.
  • AUC area under the receiver operating curve.
  • Fig. 7A – ROC plot for endpoint DGF comparing PENK and SCr at day 1, 2 and 3 post transplantation.
  • AUC area under the receiver operating curve.
  • Fig.7B – ROC plot for endpoint DGF comparing change from pre-Tx of PENK and SCr at day 1, 2 and 3 post transplantation.
  • AUC area under the receiver operating curve.
  • DGF severity groups no DGF (no RRT post transplantation); 0-1 (RRT only on day 0 or day 1); 2-7 (RRT ended between day 2 and day 7); >7 (RRT lasted longer than day 7). Cut off lines at 80 pmol/L (upper normal value) and 200 pmol/L.
  • Figure 10 A - Box and whisker plot of proenkephalin A 119-159 (PENK) by delayed graft function (DGF) defined as RRT requirement in the first seven days after transplantation, at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1). Three grey lines indicate cut-offs at 300 pmol/L, 200 pmol/L (for distinguishing DGF from no DGF) and 89 pmol/L as the upper reference limit for healthy individuals. The x-axis is log- transformed.
  • DGF delayed graft function
  • Figure 10 B Box and whisker plot of serum creatinine (SCr) by delayed graft function (DGF) defined as RRT requirement in the first seven days after transplantation, at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • DGF delayed graft function
  • a grey line indicates an SCr of 2 mg/dL for reference.
  • the x-axis is log-transformed.
  • Figure 11 Receiver operating characteristic plot for endpoint delayed graft function (DGF) defined as RRT requirement in the first seven days after transplantation, comparing change of proenkephalin A 119-159 (PENK) or serum creatinine (SCr) from 0-12 hours before transplantation to 12-24 hours after transplantation (delta d0/d1).
  • PENK proenkephalin A 119-159
  • SCr serum creatinine
  • AUC area under the curve
  • PENK proenkephalin A 119-159
  • DGF delayed (DGF), slow (SGF) and immediate (IGF) graft function at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • DGF defined as RRT in the first seven days after transplantation;
  • SGF defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 ⁇ 0.7;
  • IGF defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 > 0.7.
  • DGF defined as RRT in the first seven days after transplantation
  • SGF defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 ⁇ 0.7
  • IGF defined as no RRT in the first seven days after transplantation and quotient of SCr on day 7/SCr on day 0 > 0.7
  • a grey line indicates an SCr of 2 mg/dL for reference.
  • the x-axis is log-transformed.
  • Figure 13 A – Box and whisker plot of proenkephalin A 119-159 (PENK) by delayed graft function (DGF) severity at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • PENK proenkephalin A 119-159
  • DGF delayed graft function
  • DGF severity groups are as follows: no DGF (no RRT after transplantation); 0-1 (RRT only on day 0 or day 1); 2-7 (RRT ended between day 2 and day 7); >7 (RRT lasted longer than day 7).
  • Three grey lines indicate cut-offs at 300 pmol/L, 200 pmol/L and 89 pmol/L as the upper reference limit for healthy individuals.
  • the x-axis is log-transformed.
  • Figure 13 B Box and Whisker Plot of serum creatinine (SCr) by delayed graft function (DGF) severity at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • DGF severity groups are as follows: no DGF (no RRT after transplantation); 0-1 (RRT only on day 0 or day 1); 2-7 (RRT ended between day 2 and day 7); >7 (RRT lasted longer than day 7).
  • a grey line indicates an SCr of 2 mg/dL for reference.
  • the x-axis is log-transformed.
  • Figure 14 A - Box and whisker plot of proenkephalin A 119-159 (PENK) by reduced estimated glomerular filtration rate (eGFR) at day 30 (defined as eGFR ⁇ 30 ml/min/1.73 m2) at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • Three grey lines indicate cut-offs at 300 pmol/L, 200 pmol/L and 89 pmol/L as the upper reference limit for healthy individuals.
  • the x-axis is log-transformed.
  • Figure 14 B Box and whisker plot of serum creatinine (SCr) by reduced estimated glomerular filtration rate (eGFR) at day 30 (defined as eGFR ⁇ 30 ml/min/1.73 m2) at time points 0-12 hours before transplantation (pre-Tx) and 12-24 hours after transplantation (day 1).
  • a grey line indicates an SCr of 2 mg/dL for reference.
  • the x-axis is log-transformed.
  • Figure 15 Receiver operating characteristic plot for endpoint reduced estimated glomerular filtration rate (eGFR) at day 30 (defined as eGFR ⁇ 30 ml/min/1.73 m2) comparing change of proenkephalin A 119-159 (PENK) or serum creatinine (SCr) from 0-12 hours before transplantation to 12-24 hours after transplantation (delta d0/d1).
  • PENK proenkephalin A 119-159
  • SCr serum creatinine
  • AUC area under the curve

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Abstract

La présente invention a pour objet un procédé de diagnostic précoce et/ou de prédiction précoce d'un risque et/ou de surveillance d'un risque et/ou de prédiction de la gravité d'une fonction de greffe réduite chez un patient de transplantation rénale comprenant les étapes consistant à déterminer le niveau de pro-enképhaline ou de fragments de celle-ci dans un échantillon de fluide corporel obtenu à partir dudit patient ; et la corrélation dudit niveau de pro-enképhaline ou de fragments de celle-ci dans ledit échantillon avec le diagnostic et/ou le risque et/ou la gravité d'une fonction de greffe réduite, la fonction de greffe réduite étant une fonction de greffe lente (SGF) ou une fonction de greffe retardée (DGF) du rein.
PCT/EP2024/076971 2023-09-25 2024-09-25 Procédé de diagnostic précoce, de prédiction précoce, de surveillance ou de prédiction de la gravité d'une fonction de greffe réduite chez un patient de transplantation rénale Pending WO2025068313A1 (fr)

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